We will employ a novel approach to develop unique ALS therapeutics by focusing on drugs that activate a natural cellular protective mechanism, autophagy. Stimulating autophagy is a logical approach to treat the underlying neurodegeneration that causes ALS. It is the prime defense mechanism that neurons have for removing toxic misfolded aggregated proteins, and there is evidence that neurodegeneration of motor neurons (MNs) in ALS is caused by the buildup of toxic, misfolded, aggregated forms of SOD1, TDP43 and FUS. Mutations in these proteins cause familial ALS (fALS), and overexpression or abnormal post-translational modification of these proteins leads to MN degeneration in sporadic ALS (sALS). We will develop drugs that effectively stimulate autophagy in ALS MNs to remove the buildup of toxic proteins to block neurodegeneration and disease progression to treat sALS patients. Our collaborator, Dr. Finkbeiner, previously identified drugs that stimulate autophagy in neurons and block disease progression in murine neuronal models of ALS. He developed a pharmacophore model of neuronal autophagy inducers (NAIs) that will help us rationally design a new family of first-in-class ALS therapeutics To facilitate this discovery program, he also developed innovative models of ALS with human MNs (i-MN) derived from healthy volunteers and patients with fALS and sALS. Using an automated imaging and longitudinal analysis system called robotic microscopy (RM) he was the first to show a disease phenotype for ALS i-MNs in that they had a greater risk of death than i-MNs from healthy volunteers. This disease phenotype provides a robust, definable physiological endpoint to test new drugs for efficacy in treating ALS. We will use these novel in vitro assays with human ALS i-MNs and recently developed human i-astrocytes from patients with fALS, to develop a new family of NAIs. Using the pharmacophore model and an iterative medicinal chemistry process, Nanosyn will refine the NAI pharmacophore model to identify minimal chemical structures needed to induce autophagy. Preliminary studies have already established the feasibility of this approach by identify NAI's with increased potency and reduced safety liabilities. Nanosyn will rapidly synthesize small libraries of compounds around key chemical moieties of the model that will be screened against the human ALS i-MN models from patients with fALS and sALS. This primary screening will identify NAIs with improved potency and efficacy in promoting survival of human ALS i-MNs. Structure-activity relationship studies will further refine the model for virtual high-throughput screening to identify unique compounds that incorporate CNS-privileged pieces to select for NAIs with desired properties for treating a chronic neurodegeneration disease, such as increased CNS availability. Leads will be tested in future Phase 2 SBIR studies in vivo for inducing autophagy in the CNS by a novel approach developed by Dr. Finkbeiner that uses GFP-LC3 transgenic mice and for efficacy in SOD1-G93A transgenic mice and transgenic mice over- expressing TDP43 (A315T) in reversing motor paralysis, increasing survival, and reducing motor neuron loss.